FY2010 G0 Cell Unit

G0 Cell Unit

Abstract

The regulation of cellular proliferation and quiescence (G0 phase) in response to changes in the environment, such as the availability of nutrients, is a central issue in biology and medicine. Our research goal is to understand the molecular mechanisms by which cell division and cell cycle arrest in the G0 phase are regulated in response to available nutrients. To this end, we have adopted the fission yeast Schizosaccharomyces pombe, a simple unicellular eukaryote, as a model organism. This year, we continued to study G0 phase induced by nitrogen starvation and adaptation of S.pombe to glucose-limited condition. First, the comprehensive mutant screen was performed to identify genes, which are non-essential for cell division in the complete medium but become essential for quiescence under nitrogen starvation. Second, we analyzed previously identified genes required for G0 phase maintenance. The ubiquitin/proteasome system and autophagy cooperatively maintain mitochondria and prevent the lethal accumulation of reactive oxygen species (ROS). Finally, we investigated cell division-quiescence behavior of the fission yeast S. pombe under a wide range of glucose concentrations. The metabolomic changes under glucose-limited condition were revealed by the mass spectrometric method established in 2009. Furthermore, in collaboration with Kyoto University, regulations on mitotic chromosomes were investigated in 2010. An unexpected link between nutritional control and kinetochore function, and essential roles of Condensine throughout mitosis were reported. Comprehensive screening was continued to identify genes affecting adaptation to low glucose condition. Based on these and previopus studies, we published five original research articles and two reviews in 2010.

1. Staff

Dr. Kojiro Takeda, Group leader

Dr. Takahiro Nakamura, Researcher

Dr. Kumiko Ohta, Researcher

Dr. Kenichi Sajiki, Researcher

Dr. Takeshi Hayashi, Researcher (Kyoto)

Dr. Norihiko Nakazawa, Researcher (Kyoto)

Ms. Sakura Kikuchi, Technical staff

Ms. Risa Uehara, Technical staff

Ms. Ayaka Mori, Technical staff

Mr. Tomas Pluskal, Technical staff

Mr. Alejandro Villar-Briones, Technical staff

Ms. Tomomi Teruya, Research administrator/Secretary

Ms. Takako Shiono, Research administrator/Secretary (Kyoto)

Ms. Yuria Tahara, Research assistant

Ms. Eulaia, Lopez-Galcia, Research assistant

2. Collaborations

Theme: Metabolite structure determination using NMR

Type of collaboration: Joint research

Researchers:

Professor Masaru Ueno, Department of Molecular Biotechnology, Graduate School of Advanced Science of Matter, Hiroshima University

3. Activities and Findings

Transition from proliferation to quiescence brings about extensive changes in cellular behavior and structure. However, essential genes for quiescence establishment and maintenance are largely unknown. The fission yeast S. pombe is an excellent model for studying this problem, because it becomes quiescent under nitrogen starvation. Also in recent years, deletion mutant library sets have become commercially available (Bioneer Corp. Korea). We used the Bioneer haploid deletion set to identify genes that are non-essential for cell division in the complete medium but become essential for quiescence under nitrogen starvation. By spot testing and viability screening under nitrogen starvation, we analyzed more than 2800 deletion strains, using two laboratory automation systems to handle with the number of strains (Figure 1).

Figure 1: The strategy of screening mutants that have defects in G0 maintenance.

From these screenings, we identified 73 strains that considerably dropped the viability within 4 weeks after nitrogen starvation. The deleted genes of these strains covered cellular functions of autophagy, endosome-vacuole trafficking, exocytosis, mitochondria, phosphate signaling, chromatin remodeling, transcription factors and so on. Among them, protein phosphatase regulators are significantly abundant. Their absence caused the abnormal morphology after nitrogen starvation like septated and chitin accumulated cells and large vacuole containing cells with no polarity.

3.2 Essential Roles of the Proteasome in G0 Phase ans Proliferation

While the ubiquitin/proteasome system is pivotal to cell regulations, its essential roles in the G0 phase maintenance are elusive. As the proteasome is a target of chemical anti-cancer therapy (anti-cancer drug Velcade/Bortezomib is a specific inhibitor for the ptoteasome), it is important to deepen our knowledge on the biology of the proteasome pathway in both proliferatio and the G0 phase. Our recent study suggests that the proteasome may, in cooperation with autophagy, contribute to life span the G0 phase through quality control of mitochondria, the cellular powerhouse (Figure 2). The proteasome is important to maintain mitochondria and autophagy system scavenges damaged mitochondria, which are accelerated in ROS (reactive oxygen species) production. These results were published in Takeda et al (PNAS, 2010, original research article) and in Takeda and Yanagida (Autophagy, 2010, review article).

Figure 2: The model of cooperation of the proteasome and autophagy in G0 phase.The ubiquitin/proteasome system may be engaged in maintenance of mitochondria and autophagy scavenges damaged mitochondria in order to reduce harmful ROS accumulation.

To further our knowledge in the proteasome biology in proliferation, we aimed to identify S.pombe genes affecting the cytotoxity of anti-cancer drug Bortezomib by genome-wide synthetic lethal screening. Among the 2815 genes screened, 19 genes, whose deletions induce strong synthetic lethality with Bortezomib, were identified. We designated such genes as SLB (Synthetic Lethal with Bortezomib) genes (Table. 1). Thirteen identified genes are conserved in human and thus potentially interesting for further studies in human culture cells. If the same synthetic lethality effects occur in human cells, such SLB genes have potential for the innovation of new therapies or diagnosis. These results will be published in 2011.

3.3 S.pombe Metabolomic Analysis under Low Glucose

In the fiscal year 2010, we investigated cell division-quiescence behavior of the fission yeast S. pombe under a wide range of glucose concentrations (0-111 mM; Figure 3A). The study was published in FEBS Journal (Pluskal et al., 2011). The S. pombe cells were fixed in a microfluidic perfusion system, which was constantly supplied with fresh culture medium. The mode of cell division was surprisingly normal under highly diluted glucose concentrations (5.6 mM, 1/20 of the standard medium, within the human blood sugar levels). Division became stochastic in 2.2-4.4 mM glucose. A critical transition from division to quiescence occurred within a narrow range of concentrations (2.2-1.7 mM). Under starvation (1.1 mM), cells were mostly quiescent and only a small population of cells divided. Under fasting (0 mM) condition, division was immediately arrested with a short chronological lifespan (Figure 3B). However, when cells were first glucose starved prior to fasting, they possessed a substantially extended lifespan (~14 days).

Figure 3:A. Growth of S.pombe cells cultivated in EMM2 media with reduced glucose content. Cell length increase over time is shown. B. Viability of S.pombe cells cultivated in a medium completeklu lacking glucose, depending on the initial gkucose concentration before the medium switch.

We employed quantitative metabolomic approach for cell extracts using LC-MS, and identified specific metabolites (e.g., biotin, trehalose, ergothioneine, S-adenosyl methionine, and CDP-choline), which increased or decreased at different glucose concentrations, while nucleotide triphosphates such as ATP kept high concentrations even under starvation (Figure 4).

Figure 4: Peak areas of potential biomarker compounds in 10 different concentrations determined by the LC/MS method. Metabolite extracts were prepared three times and mean peak areas with standard deviations of the following metabolites are shown: A. ATP, ADP, AMP and adenosine. B. CDP-choline, CDP-ethanolamine. C. trehalose. D. ergothioneine. E. The time course change of the peak areas of CDP-choline and CDP-ethanolamine in cells switched to a fasting (0 mM) glucose condition from 111 mM glucose. F. The time course change of the peak areas of CDP-choline and CDP-ethanolamine in cells switched to the starvation condition (1.1 mM glucose) from 111 mM glucose.

Figure 5.A. Oxidative stress accumulation was observed using the H2DCFDA dye. B. Cells were incubated in excess (111 mM), starvation (1.1 mM) and fasting (0 mM) glucose conditions for 6 h, followed by the addition of H2O2 to the final concentration of 40 mM. }Viability (%) was measured in 20 min intervals. C. Summary of the glucose concentrations described in this manuscript. Concentrations are listed in three commonly used notations: millimolar (mM), percentage (w/v), or mg/dl. Biomarker metabolites increased (↑) or decreased (↓) in each condition are noted, as well as the corresponding cell division phenotypes.

3.4 A Possible Relationship between Kinetochore Functions and Nutritional Control

In collaboration with Kyoto University, an unexpected link between nutritional control and centromere/kinetochore functions was discovered and reported in PLoS One journal (Shiroiwa et al., 2011). The centromere is the chromosome domain on which the mitotic kinetochore forms for proper segregation. Deposition of the centromeric histone H3 (CenH3, CENP-A) is vital for the formation of centromere-specific chromatin. TheMis6-Mal2-Sim4 complex of the fission yeast S. pombe is required for the recruitment of CenH3 (Cnp1), but its function remains obscure. We performed mass spectrometric analysis of the Mis6 and Mis17 complex. The results together with the previously identified Sim4- and Mal2-TAP precipitated proteins indicated that the complex contains 12 subunits, Mis6, Sim4, Mal2, Mis15, Mis17, Cnl2, Fta1-4, Fta6-7, nine of which have human centromeric protein (CENP) counterparts. Furthermore, we found that Mis17 was highly phosphorylated (Figure 6). Domain dissection of Mis17 indicated that the carboxy-half of Mis17 is functional, while its amino-half is regulatory. Overproduction of the amino-half caused strong negative dominance, which led to massive chromosome missegregation and hypersensitivity to the histone deacetylase inhibitor TSA. Negative dominance of the Mis17 fragment is exerted while the complex and CenH3 remain at the centromere. This finding is distinct from the mislocalization or loss phenotype found in ts mis17-362 cells. Overproduction-induced negative dominance was abolished in actin- and nutrition-related kinase-deletion mutants, ssp2 (AMPK), ppk9 (AMPK), ppk15 (Yak1), ppk30 (Ark1), wis4 (Ssk2), and lsk1 (P-TEFb) (Figure 6). These findings suggest an unexpected link between Mis17 and control of the cortex actin, nutrition, and signal/transcription (Figure 6).

Figure 6: Mis17 is a regulatory module of the Mis6 complexA. Mis17 is a hyperphosphoprotein. B. The negative dominance effect of N-Mis17 overexpression was greatly diminished in six protein kinase deletion mutants, ∆ssp2, ∆ppk9, ∆ppk15, ∆ppk30, ∆lsk1, and ∆wis4.C. A cartoon for the implication of Mis17 in the Mis6-Mal2-Sim4 complex and negative dominance effect of the N-Mis17 fragment.

3.5 Roles of Condensin throughout Mitosis

Condensin is a conserved protein complex that functions in chromosome condensation and segregation. It has not been unequivocally determined whether condensin is required throughout mitosis. We critically examined whether S.pombe condensin continuously acts on chromosomes during mitosis and compared the role with that of DNA topoisomerase II (Top2). Temperature shift-up experiments were done using double mutants containing the temperature-sensitive cut14-208 (SMC2) or top2 mutations combined with the cold-sensitive mutant nda3-KM311 (b-tubulin). These experiments allowed inactivation of condensin or Top2 even after late anaphase. The results established that mitotic chromosomes require condensin and Top2 throughout mitosis, and even in telophase (Figure 7A). We then show that Cnd2/Barren is the target subunit of aurora-B like kinase Ark1. Phosphorylation sites in Cnd2 were determined by mass spectrometry and their alanine and glutamate substitutions were constructed. The Ark1-mediated phosphorylation of Cnd2 S52 occurred throughout mitosis (Figure 7B). Alanine mutants of Cnd2 exhibited broad mitotic defects, including telophase, and also severe dominant-negative effect when overproduced. By contrast, glutamate mutants alleviated segregation defect in Ark1-inhibited cells. Mutant condensin in telophase accumulated in ‘lump’ that contained telomeric DNA and proteins that failed to segregate. Condensin may thus have to keep apart the segregated chromosomes in telophase. This study was published in the Journal of Cell Science (Nakazawa et al., 2011).

Figure 7: Continuous requirement of condensin throughout mitosisA. Inactivation of condensin after entry into anaphase causes defects in telophase. The arrows indicate the lump structure. The numbers in the panels indicate time in minutes. Bar, 10 mm. B. Cnd2 S52 residue is phosphorylated throughout mitosis. The cdc25-22 Cnd2-8Myc cells were arrested at late G2 at 36°C and then released at 26°C. In contrast to the Cnd2 S52, the phosphorylation of Cut3 T19 briefly peaked only at 15 minutes, corresponding to prophase and metaphase.

3.6 Other Activitiesin Progress

Comprehensive screening mutants showing growth defect under glucose-limited condition was performed and functional analyses on isolated mutants were in progress (in collaboration with Kurume University).